WO2022136351A1 - Conjugated aflatoxin b to protect against mycotoxicosis - Google Patents

Conjugated aflatoxin b to protect against mycotoxicosis Download PDF

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WO2022136351A1
WO2022136351A1 PCT/EP2021/086953 EP2021086953W WO2022136351A1 WO 2022136351 A1 WO2022136351 A1 WO 2022136351A1 EP 2021086953 W EP2021086953 W EP 2021086953W WO 2022136351 A1 WO2022136351 A1 WO 2022136351A1
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afb
conjugated
animal
don
animals
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PCT/EP2021/086953
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English (en)
French (fr)
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Maarten Hendrik Witvliet
Ruud Philip Antoon Maria Segers
Mateusz Walczak
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Intervet International B.V.
Intervet Inc.
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Priority to EP21843937.0A priority Critical patent/EP4267174A1/en
Priority to US18/257,600 priority patent/US20240024446A1/en
Priority to JP2023537667A priority patent/JP2023554139A/ja
Priority to CN202180087198.XA priority patent/CN116615225A/zh
Priority to CA3202680A priority patent/CA3202680A1/en
Priority to AU2021405239A priority patent/AU2021405239A1/en
Priority to MX2023007625A priority patent/MX2023007625A/es
Publication of WO2022136351A1 publication Critical patent/WO2022136351A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0013—Therapeutic immunisation against small organic molecules, e.g. cocaine, nicotine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/38—Albumins
    • A61K38/385—Serum albumin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6081—Albumin; Keyhole limpet haemocyanin [KLH]

Definitions

  • the invention in general pertains to protection of animals against mycotoxicosis induced by mycotoxins.
  • the invention pertains to protection against mycotoxicosis induced by aflatoxin B (AFB).
  • Aflatoxins produced by the Aspergillus species are highly toxic, carcinogenic, and cause severe contamination to food sources, leading to serious health consequences. Contaminations by aflatoxins have been reported in food and feed, such as groundnuts, millet, sesame seeds, maize, wheat, rice, fig, spices and cocoa due to fungal infection during pre- and post-harvest conditions. Besides these food products, commercial products like peanut butter, cooking oil and cosmetics have also been reported to be contaminated by aflatoxins. Even a low concentration of aflatoxins is hazardous for human and livestock.
  • AFs aflatoxins
  • Aflatoxins are chemically difuranocoumarin derivatives with a bifuran group attached to the coumarin nucleus and a pentanone ring (in case of AFBs) or a lactone ring (in case of AFGs).
  • AFB’s are generally more toxic than AFGs.
  • the two major AFBs among the identified 20 AFs are the closely related AFB1 and AFB2.
  • AFB1 is a potent carcinogen to humans, and is associated with serious health complications after ingestion of AFB contaminated food. It has been a causal factor for liver cancer and acute hepatitis as well as periodic outbreaks of acute aflatoxicosis leading to death.
  • AFB induced mycotoxicosis
  • AFB also induces disease in all sorts of livestock animals, although dairy and beef cattle seem to be somewhat are more susceptible to aflatoxine induced mycotoxicosis (also denoted as aflatoxicosis) than other species such as swine and poultry. Young animals of all species are more susceptible to the effects of aflatoxins than mature animals. Pregnant and growing animals are less susceptible than young animals but more susceptible than nature animals.
  • Toxicity due to AFBs under natural conditions, is usually sub-acute or chronic, depending on the level of exposure. Occasionally, acute cases are also seen. In general, affected animals show reduced growth rate, weight loss, immune suppression, icterus, hemorrhagic enteritis, reduced performance, and ultimately death.
  • fungi can produce mycotoxins and organic chemicals that are responsible for various toxic effects referred to as mycotoxicosis.
  • This disease is caused by exposure to mycotoxins, pharmacologically active compounds produced by filamentous fungi contaminating foodstuffs or animal feeds.
  • Mycotoxins are secondary metabolites not critical to fungal physiology, that are extremely toxic in minimum concentrations to vertebrates upon ingestion, inhalation or skin contact. About 400 mycotoxins are currently recognized, subdivided in families of chemically related molecules with similar biological and structural properties.
  • mycotoxins of greatest public interest and agroeconomic significance include aflatoxins (AF), ochratoxins (OT), trichothecenes (T; including deoxynivalenol, abbreviated DON), zearalenone (ZEA), fumonisin (F), tremorgenic toxins, and ergot alkaloids.
  • Mycotoxins have been related to acute and chronic diseases, with biological effects that vary mainly according to the diversity in their chemical structure, but also with regard to biological, nutritional and environmental factors.
  • mycotoxicosis The pathophysiology of mycotoxicosis is the consequence of interactions of mycotoxins with functional molecules and organelles in the animal cell, which may result in carcinogenicity, genotoxicity, inhibition of protein synthesis, immunosuppression, dermal irritation, and other metabolic perturbations. In sensitive animal species, mycotoxins may elicit complicated and overlapping toxic effects. Mycotoxicosis are not contagious, nor is there significant stimulation of the immune system. T reatment with drugs or antibiotics has little or no effect on the course of the disease. To date no human or animal vaccine is available for combating mycotoxicosis.
  • mycotoxins do not need the involvement of the toxin producing fungus and are considered as abiotic hazards, although with biotic origin.
  • mycotoxicosis have been considered examples of poisoning by natural means, and protective strategies have essentially focused on exposure prevention. Human and animal exposure occurs mainly from ingestion of the mycotoxins in plant-based food.
  • mycotoxins Metabolism of ingested mycotoxins could result in accumulation in different organs or tissues; mycotoxins can thus enter into the human food chain through animal meat, milk, or eggs (carry over). Because toxigenic fungi contaminate several kinds of crops for human and animal consumption, mycotoxins may be present in all kinds of raw agricultural materials, commodities and beverages.
  • the Food and Agriculture Organization (FAO) estimated that 25% of the world’s food crops are significantly contaminated with mycotoxins.
  • the best strategies for mycotoxicosis prevention include good agricultural practice to reduce mycotoxins production on crop and control programs of food and feed commodities to ensure that mycotoxin levels stand below predetermined threshold limits. These strategies may limit the problem of contamination of commodities with some groups of mycotoxins with high costs and variable effectiveness.
  • mycotoxins are low molecular weight, usually non-proteinaceous molecules, which are not ordinarily immunogenic (haptens), but can potentially elicit an immune response when attached to a large carrier molecule such as a protein.
  • Methods for conjugation of mycotoxins to protein or polypeptide carrier and optimization of conditions for animal immunization have been extensively studied, with the purpose of producing monoclonal or polyclonal antibodies with different specificities to be used in immunoassay for screening of mycotoxins in products destined for animal and human consumption.
  • mycotoxin vaccination would thus be based on generating antibodies against the mycotoxoid with an enhanced ability to bind native mycotoxin compared with cellular targets, neutralizing the toxin and preventing disease development in the event of exposure.
  • a potential application of this strategy has been demonstrated in the case of mycotoxins belonging to the AF group (Giovati et al, 2015), but not for any of the other mycotoxins.
  • the protective effect has not been demonstrated against mycotoxicosis of the vaccinated animal as such, but only against carry over in dairy cows to their milk, so as to protect people that consume the milk or products made thereof from mycotoxicosis.
  • conjugated aflatoxin is suitable for use in a method to actively protect an animal against AFB induced mycotoxicosis. It was found that there was no need to convert the AFB into a toxoid, the conjugated toxin appeared to be safe for the treated host animal. Also, it was surprising to see that an immune response induced against a small molecule such as a mycotoxin is, is strong enough to protect the animal itself against mycotoxicosis after ingestion of the mycotoxin post treatment. Such actual protection of an animal by inducing in that animal an active immune response against a mycotoxin itself has not been shown in the art for any mycotoxin.
  • AFB conjugated aflatoxin
  • Mycotoxicosis is the disease resulting from exposure to a mycotoxin.
  • the clinical signs, target organs, and outcome depend on the intrinsic toxic features of the mycotoxin and the quantity and length of exposure, as well as the health status of the exposed animal.
  • To protect against mycotoxicosis means to prevent or decrease one or more of the negative physiological effects of the mycotoxin in the animal, such as a decrease in average daily weight gain.
  • Aflatoxin B denotes aflatoxins that are chemically difuranocoumarin derivatives with a bifuran group attached to the coumarin nucleus and a pentanone ring.
  • AFB1 and AFB2 covers AFB1 and AFB2.
  • the chemical formula of AFB1 is C17H12O6 (cas no 1162-65-8) and that of AFB2 is C17H14O6 (cas no 7220-81-7), having no double bond in the bifuran group.
  • the structural formula of AFB1 is given here below.
  • a conjugated molecule is a molecule to which an immunogenic compound is coupled through a covalent bond.
  • the immunogenic compound is a large protein such as KLH, BSA or OVA.
  • An adjuvant is a non-specific immunostimulating agent.
  • each substance that is able to favor or amplify a particular process in the cascade of immunological events, ultimately leading to a better immunological response i.e. the integrated bodily response to an antigen, in particular one mediated by lymphocytes and typically involving recognition of antigens by specific antibodies or previously sensitized lymphocytes
  • an adjuvant is in general not required for the said particular process to occur, but merely favors or amplifies the said process.
  • Adjuvants in general can be classified according to the immunological events they induce. The first class, comprising i.a.
  • ISCOM immunological complexes
  • saponins or fractions and derivatives thereof such as Quil A
  • aluminum hydroxide liposomes
  • cochleates polylactic/glycolic acid
  • APC antigen presenting cells
  • the second class comprising i.a. oil emulsions (either W/O, O/W, W/O/Wor O/W/O), gels, polymer microspheres (Carbopol), non-ionic block coplymers and most probably also aluminum hydroxide, provide for a depot effect.
  • the third class comprising i.a.
  • CpG-rich motifs monophosphoryl lipid A, mycobacteria (muramyl dipeptide), yeast extracts, cholera toxin, is based on the recognition of conserved microbial structures, so called pathogen associated microbial patterns (PAMPs), defined as signal 0.
  • PAMPs pathogen associated microbial patterns
  • the fourth class comprising i.a. oil emulsion surface active agents, aluminum hydroxide, hypoxia, is based on stimulating the distinguishing capacity of the immune system between dangerous and harmless (which need not be the same as self and non-self).
  • the fifth class comprising i.a. cytokines, is based on upregulation of costimulatory molecules, signal 2, on APCs.
  • a vaccine is in the sense of this invention is a constitution suitable for application to an animal, comprising one or more antigens in an immunologically effective amount (i.e. capable of stimulating the immune system of the target animal sufficiently to at least reduce the negative effects of a challenge with a disease inducing agent, typically combined with a pharmaceutically acceptable carrier (i.e. a biocompatible medium, viz.
  • a medium that after administration does not induce significant adverse reactions in the subject animal, capable of presenting the antigen to the immune system of the host animal after administration of the vaccine such as a liquid containing water and/or any other biocompatible solvent or a solid carrier such as commonly used to obtain freeze- dried vaccines (based on sugars and/or proteins), optionally comprising immunostimulating agents (adjuvants), which upon administration to the animal induces an immune response for treating a disease or disorder, i.e. aiding in preventing, ameliorating or curing the disease or disorder.
  • Active protection induced by a vaccine is protection of a vaccinated subject itself by induction of antibodies by the vaccine in the subject, which antibodies protect the subject against a later challenge with the corresponding disease causing pathogen or compound.
  • Active protection is opposed to passive protection whereby a subject animal receives ready-made antibodies produced outside of its body (e.g. in a laboratory animal, or by a mother animal, or recombinantly), in order to protect against the corresponding disease causing pathogen or compound.
  • the conjugated AFB is systemically administered to the animal.
  • local administration for example via mucosal tissue in the gastro-intestinal tract (oral or anal cavity) or in the eyes (for example when immunising chickens) is known to be an effective route to induce an immune response in various animals
  • systemic administration leads to an adequate immune response for protecting animals against a AFB induced mycotoxicosis. It was found in particular that effective immunisation can be obtained upon intramuscular, oral and/or intradermal administration.
  • the age of administration is not critical, although it is preferred that the administration takes place before the animal is able to ingest feed contaminated with substantial amounts of AFB. Hence a preferred age at the time of administration of 6 weeks or younger. Further preferred is an age of 4 weeks or younger, such as for example an age of 1-3 weeks.
  • the conjugated AFB is administered to the animal at least twice.
  • many animals in particular swine chickens, ruminants
  • the immune system of the animals will not be triggered to produce anti-AFB antibodies by natural exposure to AFB, simply because naturally occurring AFB is not immunogenic. So, the immune system of the animals is completely dependent on the administration of the conjugated AFB.
  • the time between the two shots of the conjugated AFB can be anything between 1 week and 1-2 years.
  • a regime of a prime immunisation for example at 1-3 weeks of age, followed by a booster administration 1-4 weeks later, typically 1-3 weeks later, such as 2 weeks later, will suffice.
  • Older animals may need a booster administration every few months (such as 4, 5, 6 months after the last administration), or on a yearly or biannual basis as is known form other commercially applied immunisation regimes for animals.
  • the conjugated AFB is used in a composition comprising an adjuvant in addition to the conjugated AFB.
  • An adjuvant may be used if the conjugate on itself is not able to induce an immune response to obtain a predetermined level of protection.
  • conjugate molecules are known that are able to sufficiently stimulate the immune system without an additional adjuvant, such as KLH or BSA, it may be advantageous to use an additional adjuvant. This could take away the need for a booster administration or prolong the interval for the administration thereof. All depends on the level of protection needed in a specific situation.
  • a type of adjuvant that was shown to be able and induce a good immune response against AFB when using conjugated-AFB as immunogen is an emulsion of water and oil, such as for example a water-in-oil emulsion or an oil-in-water emulsion.
  • the former is typically used in poultry while the latter is typically used in animals who are more prone to adjuvant induced site reactions such as swine and ruminants.
  • the conjugated AFB comprises AFB conjugated to a protein having a molecular mass above 10.000 Da.
  • proteins in particular keyhole limpet hemocyanin (KLH) and ovalbumin (OVA), have been found to be able and induce an adequate immune response in animals, in particular in swine and chickens.
  • KLH keyhole limpet hemocyanin
  • OVA ovalbumin
  • a practical upper limit for the protein might be 100 MDa.
  • the animal is believed to be protected against a decrease in average daily weight gain, immune suppression, icterus, hemorrhagic enteritis as a result of the ingestion of AFB, thus one or more of these signs of mycotoxicosis induced by AFB.
  • the objective of this study was to evaluate the efficacy of conjugated deoxynivalenol to protect an animal against mycotoxicosis due to DON ingestion.
  • pigs were immunised twice with DON-KLH before being challenged with toxic DON.
  • Different routes of immunisation were used to study the influence of the route of administration.
  • Group 1 was immunised intramuscularly (IM) at both ages.
  • Group 2 received an IM injection at one week of age and an oral boost at three weeks of age.
  • Group 3 was immunised intradermally (ID) two times. From 51 weeks of age groups 1-3 were challenged during 4 weeks with DON administered orally in a liquid.
  • Group 4 was not immunised but was only challenged with DON as described for groups 1-3.
  • Group 5 served as a control and only received a control fluid, from the age of 5.5 weeks for 4 weeks.
  • the DON concentration in the liquid formulation corresponded to an amount of 5.4 mg/kg feed. This corresponds to an average amount of 2.5 mg DON per day.
  • the DON concentration in the liquid formulation corresponded to an amount of 5.4 mg/kg feed. This corresponds to an average amount of 2.5 mg DON per day.
  • Test Article 1 comprising DON-KLH at 50 pg/ml in an oil-in-water emulsion for injection (X-solve 50, MSD AH, Boxmeer) which was used for IM immunization;
  • Test Article 2 comprising DON-KLH at 50 pg/ml in a water-in-oil emulsion (GNE, MSD AH, Boxmeer) which was used for oral immunization
  • Test Article 3 comprising DON-KLH at 500 pg/ml in an oil-in-water emulsion for injection (X-solve 50) for ID immunisation.
  • the challenge deoxynivalenol (obtained from Fermentek, Israel) was diluted in 100 % methanol at a final concentration of 100 mg/ml and stored at ⁇ -15 °C. Prior to usage, DON was further diluted and supplied in a treat for administration.
  • Table 1 1gG titres As depicted in Table 2 all immunised animals, including the animals in Group 2 that showed no significant anti-DON IgG titre increase, showed a significant higher weight gain during the first 15 days compared to the challenge animals. With respect to the challenged animals, all animals gained more weight over the course of the study.
  • the condition of the small intestines was also monitored.
  • table 3 the villus/crypt ratio is depicted.
  • the animals in group 3 had an average villus crypt/crypt ratio comparable to the healthy controls (group 5), while the non-immunised, challenged group (group 4) had a much lower (statistically significant) villus crypt ratio.
  • group 1 and group 2 had a villus/crypt ratio which was significantly better (i.e. higher) compared to the nonimmunised challenge control group. This indicates that the immunisation protects against the damage of the intestine, initiated by DON.
  • Table 3 villus/crypt ratio
  • the general condition of other organs was also monitored, more specifically the liver, the kidneys and the stomach. It was observed that all three test groups (groups 1-3) were in better health than the non-immunised challenge control group (group 4).
  • group 4 a summary of the general health data is depicted.
  • the degree of stomach ulcer is reported from - (no prove of ulcer formation) to ++ (multiple ulcers).
  • the degree of stomach inflammation is reported from - (no prove of inflammation) to ++/- (initiation of stomach inflammation).
  • the objective of this study was to evaluate the effects of immunization with a DON conjugate on the toxicokinetics of DON ingestion. To examine this, pigs were immunised twice with DON-KLH before being fed toxic DON.
  • mice Ten 3 week old pigs were used in the study, divided over 2 groups of 5 pigs each.
  • the pigs in Group 1 were immunised IM twice at 3 and 6 weeks of age with DON-KLH (Test Article 1; examplel).
  • Group 2 served as a control and only received a control fluid.
  • the animals were each administered DON (Fermentek, Israel) via a bolus at a dose of 0.05 mg/kg which (based on the daily feed intake) resembled a contamination level of 1 mg/kg feed.
  • Blood samples of the pigs were taken juts before DON administration and 0.25, 0.5, 0.75, 1 , 1.5, 2, 3, 4, 6, 8, and 12 h post DON administration.
  • Plasma analysis of unbound DON was done using a validated LC-MS/MS method on an Acquity® LIPLC system coupled to a Xevo® TQ-S MS instrument (Waters, Zellik, Belgium).
  • the lower limit of quantification of DON in pig plasma using this method is 0.1 ng/ml.
  • Toxicokinetic modeling of the plasma concentration-time profiles of DON was done by noncompartmental analysis (Phoenix, Pharsight Corporation, USA). Following parameters were calculated: area under the curve from time zero to infinite (AUCo ⁇ ), maximal plasma concentration (Cmax), and time at maximal plasma concentration (tmax).
  • the objective of this study was to evaluate the efficacy of different conjugated deoxynivalenol products.
  • Blood sampling took place at day 0 and 14, as well as on day 35, 56, 70 and 84. Serum was isolated for the determination of IgY against DON. At day 0 and 14 blood samples were isolated just before immunisation.
  • the conjugated DON also induces an anti-DON titre in chickens.
  • GNE adjuvant increases the response substantially but appears to be not essential for obtaining a net response as such.
  • Example 5 Serological response against AFB conjugate in swine
  • the aim of this experiment was to assess whether or not the use of conjugated AFB in a vaccine can induce antibodies against aflatoxin in vaccinated swine.
  • the vaccine contained Aflatoxin B1 (AFB1) conjugated to Bovine Serum Albumin (BSA).
  • BSA Bovine Serum Albumin
  • the conjugate was mixed with a mineral oil-containing adjuvant (XSolve 50) at a final concentration of 50 pg/ml, and applied by intramuscular (IM) administration.
  • IM intramuscular
  • 2 groups of 6 animals were used at three weeks of age. Group one received a PCV vaccine, Porcilis® PCV (as a negative control) and Group 2 the AFB1- BSA vaccine. All primes were at three weeks of age and the boosters were at seven weeks of age. The animals were monitored for 12 weeks after start of the study.
  • Blood sampling took place at day 0 and 14, as well as on day 35, 56, 70 and 84. Serum was isolated for the determination of IgY against AFB1. At day 0 and 14 blood samples were isolated just before immunisation.
  • the conjugated AFB1 induces an anti-AFB1 titre in chickens.
  • GNE adjuvant increases the response substantially but appears to be not essential for obtaining a net response as such.
  • the objective of this study was to evaluate the serological response to AFB1-KLH in fish (Tilapia; Oreochromis sp).
  • tilapia fish were used (weighing on average 20 g), divided into two groups of fifty fish.
  • the first group was injected intraperitoneally (IP) with 0.05ml an AFB1-KLH mycotoxin vaccines in GNE adjuvant.
  • the AFB1-KLH was present at a final concentration of 12.5 pg per dose.
  • the second group was injected with standard vaccine dilution buffer (SVDB) to serve as a negative control.
  • SVDB standard vaccine dilution buffer
  • the conjugated AFB1 induces an anti-AFB1 titre in the fish.
  • Example 7 an in vitro potency test / neutralization assay was performed with the sera raised as described in Example 8. For this, C6 cells were seeded at 2.0*10 4 cells/ml, 100 pl per well, and grown at 37°C, 5% CO2 for 3 days. Cells were incubated for 48h, with a combination of 20 pg/ml AFB1 and 32x diluted serum derived from fish vaccinated with AFB1-KLH in GNE or vaccinated with a placebo. Percentages of live cells were determined by microscopical evaluation.
  • Serum from fish vaccinated with AFB1-KLH in GNE contains antibodies against AFB1 (see Example 8) and appeared to be protected against AFB1 damage in C6 cells.
  • Serum from fish vaccinated with placebo did not contain antibodies against AFB1 (see Example 8) and did not protect against AFB1 damage.

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PCT/EP2021/086953 2020-12-22 2021-12-21 Conjugated aflatoxin b to protect against mycotoxicosis WO2022136351A1 (en)

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EP21843937.0A EP4267174A1 (en) 2020-12-22 2021-12-21 Conjugated aflatoxin b to protect against mycotoxicosis
US18/257,600 US20240024446A1 (en) 2020-12-22 2021-12-21 Conjugated aflatoxin b to protect against mycotoxicosis
JP2023537667A JP2023554139A (ja) 2020-12-22 2021-12-21 マイコトキシン症から保護するためのコンジュゲート化アフラトキシンｂ
CN202180087198.XA CN116615225A (zh) 2020-12-22 2021-12-21 防护霉菌毒素中毒的缀合黄曲霉毒素b
CA3202680A CA3202680A1 (en) 2020-12-22 2021-12-21 Conjugated aflatoxin b to protect against mycotoxicosis
AU2021405239A AU2021405239A1 (en) 2020-12-22 2021-12-21 Conjugated aflatoxin b to protect against mycotoxicosis
MX2023007625A MX2023007625A (es) 2020-12-22 2021-12-21 Aflatoxina b conjugada para proteger contra micotoxicosis.

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